ABSTRACT Cancer is a heterogeneous disease originating from an accumulation of genetic and epigenetic mutations in a single normal cell. Precise characterization of a broad spectrum of heterogeneous populations of tumor cells within individual patients at different molecular levels would greatly facilitate understanding of tumor initiation, progression, metastasis and therapeutic response with the potential to move toward precision medicine. With recent advances in antibody-based flow cytometry and mass cytometry, high-throughput targeted proteomics analysis of single cells has become possible. However, they share common shortcomings with other antibody-based methods, and lack quantitation accuracy to provide absolute protein amounts or concentrations. Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative in terms of its being antibody-free, high multiplex, and high precision/accuracy. However, there are two major technical challenges for targeted MS analysis of single cells: 1) effective processing of single cells, and 2) sufficient MS sensitivity. To address these two challenges, we propose to develop an ultrasensitive targeted MS system for enabling rapid, comprehensive, precise analysis of single cells. The new development is built upon 1) incorporation of a new `carrier protein' concept into our simplified nano-proteomics preparation platform (SNaPP) for automated robust processing of single cells, and 2) leveraging disruptive MS technologies pioneered at Pacific Northwest National Laboratory with effective integration of high-efficiency SPIN (sub- ambient pressure ionization with nanoelectrospray) source and ultrafast high-resolution SLIM (structures for lossless ion manipulation)-based ion mobility separation with a state-of-the-art high resolution/ sensitivity time-of-flight (TOF) MS through electrodynamic ion funnel interfaces for high-efficiency ion transmission. When coupling with ultralow-flow high-resolution reversed-phase liquid chromatography (LC), the new MS platform is expected to provide >50 and ~100-fold improvement in sensitivity and sample throughput, respectively (i.e., ~0.2-2 zmols or ~120-1200 molecules of sensitivity and ~5-10 mins per sample) when compared to standard targeted MS platforms. In combination with cSNaPP for effective processing of single cells, such levels of improvement could allow the new LC-MS platform for precise quantification of the majority of the entire proteome in single cells and ~150 samples per day. We anticipate that the new MS system will become a convenient indispensable quantitation tool for routine proteomics analysis of single cells and make substantial contributions to current biomedical research.